The TP53 gene encodes the tumor suppressor protein 53, which is critical for maintaining genomic stability and preventing tumorigenesis. Mutations in TP53, particularly missense mutations, have a substantial impact on cancer progression because they give gain-of-function features that enhance proliferation, metastasis, and treatment resistance. This review examines the mechanisms underlying p53 mutations, including their interactions with critical regulatory circuits, and assesses novel medication and prodrug options targeting TP53 mutations in various malignancies. Small-molecule correctors, statins, Hsp90 inhibitors, and new drugs like Eprenetapopt and COTI-2 are among the therapeutic options proposed. The mechanisms of action and potential efficacy in treating leukemia, lung, breast, and ovarian malignancies are investigated. Emerging techniques for restoring wild-type p53 functionality or degrading mutant p53 demonstrate the therapeutic potential of these approaches. Challenges such as medication resistance, side effects, and the chemical complexity of p53 pathways are also addressed, emphasizing the importance of ongoing research. This review contributes to our understanding of TP53-targeted cancer medicines, offering hope for more innovative treatments with improved outcomes.

The BTK inhibitor ibrutinib and the BCL-2 antagonist venetoclax are active therapies as single agents in Waldenström macroglobulinemia (WM). In this phase 2 study, we sought to investigate the combination of ibrutinib and venetoclax as a 2-year fixed-duration, oral-based, chemotherapy-free regimen in symptomatic, treatment-naive WM patients. All patients had MYD88 mutations, 17 (38%) had CXCR4 mutations, and 4 (9%) had TP53 alterations. Following enrollment of 45 patients, treatment and enrollment were stopped due to the occurrence of ventricular arrhythmias in 4 (9%), which included two grade 5 events. The median treatment time was 10 cycles, each lasting 28 days. Follow-up continued after protocol treatment was terminated. The very good partial response/complete response (VGPR/CR) rate was 42%, and it was lower in patients withCXCR4 (29% vs. 50%) or TP53 (25% vs. 44%) mutations. With a median follow-up of 49 months, the median progression-free survival (PFS) was 36 months (range 28-42). The median treatment-free survival (TFS) and overall survival (OS) werenot reached, and the 4-year TFS and OS rates were 73% and 91%, respectively. The median PFS after end of therapy (PFS-EOT) was 29 months. TP53 mutations were associated with inferior PFS, TFS, and PFS-EOT, while CXCR4 mutations did not adversely impact these outcomes. Given the deep and durable responses seen in this study, concurrent BTK and BCL-2 inhibition warrants further development in WM. TP53 mutations emerged as an adverse factor in WM in this combination study.

Malignant germ cell tumors (GCTs) are relatively rare tumors with limited therapeutic options. This study examined the homologous recombination deficiency (HRD) status and genomic characteristics in 14 patients with GCT who underwent next-generation sequencing. HRD was evaluated using the genomic instability score (GIS), which incorporates three components: loss of heterozygosity (LOH), telomeric allelic imbalance (TAI), and large-scale transition (LST). Our findings showed that 57.1% (8/14) of participants were HRD positive. Female patients presented a significantly higher prevalence of HRD positivity (87.5%, 7/8) compared with male patients (16.7%, 1/6). HRD positivity in female cases had a mean GIS of 56 (range 48-76), including one patient harboring a germline BRCA2 p.S2670L (c.8009C>T) likely pathogenic mutation. LST demonstrated the strongest correlation with the integrated GIS (R2 = 0.945), followed by LOH (R2 = 0.872). Distinct mutation patterns were observed based on gender. GCTs in male cases predominantly exhibited mutations in TP53 (50% in yolk sac tumors; 50% in teratomas). GCTs in male cases also demonstrated TP53 mutations in one mediastinal yolk sac tumor and one mediastinal teratoma. Male cases showed recurrent alterations in KRAS, PRKDC, and CDKN1B, alongside frequent chromosome 12p amplifications in two cases. One particularly complex mediastinal teratoma in a male patient, featuring rhabdomyosarcomatous transformation, displayed bidirectional intergenic (TWSG1, MANEA-DT)-ROS1fusions, a HRAS-intergenic (RNH1) fusion, and MET focal amplification. These findings suggest a promising therapeutic opportunity with poly (ADP-ribose) polymerase (PARP) inhibitors for female patients with HRD-positive GCTs. Additionally, the complex composition of gene variants found in male patients with GCTs, including ROS1 fusions and MET focal amplification, points toward potential targeted therapeutic strategies. This study underscores the presence of sex-specific therapeutic vulnerabilities in GCTs, which warrant further exploration in larger cohorts. © 2026 The Pathological Society of Great Britain and Ireland.

Bisphenol A (BPA), a ubiquitous environmental endocrine disruptor, has been implicated in carcinogenesis; however, its effects on thyroid cells remain unclear. This study investigated BPA-induced cytotoxicity and genotoxicity in thyroid cell lines, including normal follicular (Nthy-ori 3-1), papillary carcinoma (TPC-1 and BCPAP) and anaplastic carcinoma (8505C) cells, including exposure to the specific migration limit (SML) established by Brazilian regulations (1 μg/mL). Cells were exposed for 24 and 48 h, and membrane integrity, metabolic activity, and DNA damage were assessed using trypan blue exclusion, CCK-8, and comet assays, respectively. Nthy-ori 3-1 exhibited non-monotonic cytotoxicity, with 60% mortality at the SML (24 h). The 8505C line displayed notable resistance to membrane damage at high concentrations but was highly sensitive to genotoxicity, which intensified after BPA exposure and may be related to TP53 mutations. The SML dose caused significant DNA damage in normal cells, whereas papillary lines were less affected. These findings demonstrate cell-context-dependent BPA effects and suggest that current regulatory thresholds may not adequately protect thyroid health, particularly in individuals with pre-existing thyroid conditions. BPA's impact involves both metabolic disruption and impaired DNA repair pathways, supporting its potential carcinogenicity.

Medulloblastoma (MB) is a biologically and clinically heterogeneous pediatric brain tumor. However, large-scale molecular subgrouping studies have mainly been conducted in Western populations, and comprehensive data from Asia are limited. To address this gap, we analyzed 242 MB cases collected from 39 institutions through the Japan Pediatric Molecular Neuro-Oncology Group, performing centralized molecular classification using NanoString-based gene expression profiling, DNA methylation arrays, and multiplex ligation-dependent probe amplification (MLPA)-based copy number profiling, supplemented by targeted sequencing. The subgroup distribution was 16.1% WNT, 24.8% SHH, 17.4% Group 3, and 41.7% Group 4. CTNNB1 mutations and monosomy 6 characterized all WNT cases, whereas MYCN amplification and TP53 mutations were independent adverse markers in SHH MB. Group 3 showed the worst survival, with MYC amplification and metastasis as poor prognostic factors. In Group 4, large cell/anaplastic histology predicted poor outcomes, whereas chromosome 11 loss was correlated with a favorable prognosis. Whole chromosomal aberration-defined favorable-risk patterns consistently indicate improved outcomes in non-WNT/non-SHH MBs. We also developed a simplified MLPA-based classifier targeting six loci on chromosomes 7, 8, and 11 (SEE-6-CNA), which enabled robust and clinically feasible prognostic stratification. Overall, our findings confirm that the molecular subgroup-specific features of Japanese MBs are largely concordant with global observations and that SEE-6-CNA provides a cost-effective tool to support individualized treatment planning, particularly in resource-limited settings.

Esophageal adenocarcinoma (EAC), the dominant subtype of esophageal cancer in developed countries, is a growing health problem, characterized by poor patient prognosis and dismal survival due to ineffective screening tools and a lack of efficacious options targeting the interception or treatment of EAC. Despite molecular advances, molecular targeting of EAC remains elusive, suggesting the need for identifying alternative targets with improved prognostic and therapeutic value. Herein, we performed RNA-sequencing analysis in EAC and Barrett's Esophagus (BE) precursor lesions to identify isoform switching events significantly linked with all-cause and cancer-specific mortality. Patients were stratified based on histopathology alone or in combination with TP53 mutation status, the most commonly mutated gene in EAC. To gain mechanistic insight, we performed isoform-specific siRNA knockdown of two isoforms, TTLL12 and HM13, both linked to patient survival, and investigated mechanisms associated with isoform dysregulation and whether targeting specific isoforms in EAC acts synergistically to improve therapeutic potential. Isoform-specific knockdown of TTLL12 and HM13 significantly decreased the viability of two EAC cell lines, sensitized EAC cell lines to standard-of-care chemotherapy agents (paclitaxel and carboplatin) with synergy, and inhibited EAC cell migratory potential. Knockdown of the TTLL12 isoform led to activation of chaperone-mediated autophagy, which, in turn, decreased expression of CHK1 and TP53; whereas knockdown of the HM13 isoform activated the unfolded protein response and induced endoplasmic reticulum stress-induced autophagy and apoptosis. In addition, HM13 isoform knockdown increased the response to an anti-PD-L1 agent, avelumab, in EAC cells, suggesting a role for isoform switching in immunosuppression. Taken together, study results suggest that isoform switching may provide novel insight for the identification of prognostic markers and inform new potential therapeutic targets for EAC treatment or prevention.

Small cell lung carcinoma (SCLC) is classically defined by biallelic inactivation of RB1 and TP53. However, a small subset of tumors retains Rb expression and exhibits distinct molecular features. Here, we report two Rb-retained SCLC cases that expand the biological and therapeutic spectrum of this subgroup. Both tumors occurred in middle-aged women, showed small cell morphology with some variant features, and displayed complex copy number alterations. Case 1 harbored a truncal KRAS p.G12C mutation with high-level amplification of chromosome 11q13-q14, including CCND1, and demonstrated a clinical response to sotorasib. Case 2 harbored a TP53 mutation, CDKN2A loss, STK11 inactivation, and a novel IKZF2::ERBB4 fusion. These findings highlight the molecular heterogeneity of Rb-retained SCLC and demonstrate that this subgroup can harbor clinically actionable oncogenic drivers. Accordingly, routine assessment of Rb expression in SCLC, followed by comprehensive molecular profiling of Rb-retained tumors, is warranted to uncover therapeutically relevant targets.

TP53 and PPM1D are key regulators of DNA damage response and repair, and somatic mutations in these genes often co-occur in hematopoietic cells, expanding under genotoxic stress. Unlike TP53 mutations, where mechanisms of progression are defined, pathways underlying clonal fitness and transformation in PPM1D mutant cells remain unclear. In collaboration with 5 academic institutions, we analyzed the clinical and molecular landscape of 337 patients with clonal hematopoiesis (CH) and clonal cytopenia of undetermined significance (CCUS) across 4 genotypes: PPM1Dmt/TP53wt (n = 170 [50%]), PPM1Dmt/TP53mt (n = 25 [7%]), TP53mt/PPM1Dwt (n = 17 [5%]), and TP53wt/PPM1Dwt (n = 125 [38%]). All PPM1D variants were truncating, located in exon 6 of the gene, with a median variant allele frequency (VAF) of 6% (range, 0.3%-64%). The PPM1Dmt/TP53mt genotype was most frequently encountered in therapy-related CH/CCUS (t-CH/t-CCUS; 80%, 66.5%, 76.5%, and 19%; P ≤ .001) and had a shorter time interval to detection from last genotoxic exposure (6.2, 5.9, 11.25, and 24.5 months; P ≤ .001) compared with PPM1Dmt/TP53wt, TP53mt/PPM1Dwt, and TP53wt/PPM1Dwt genotypes, respectively. Acknowledging the short follow-up duration, rates of malignant transformation were lower in the PPM1Dmt/TP53wt (2%) and PPM1Dmt/TP53mt (4%) groups compared with PPM1Dwt/TP53wt (12%) and PPM1Dwt/TP53mt (17%) groups (P ≤ .001), respectively. In summary, PPM1D mutations are frequently observed in t-CH/t-CCUS, with low median VAFs, and are associated with low rates of progression, even when comutated with TP53.

To improve the tolerability of posttransplant maintenance and outcomes despite poor-risk disease genetics, we conducted a phase 1 study of venetoclax (Ven) with FluBu2 (fludarabine/busulfan) reduced-intensity chemotherapy transplantation using tacrolimus/methotrexate graft-versus-host disease (GVHD) prophylaxis, followed by all-oral Ven/decitabine-cedazuridine (Ven/Dec-c) maintenance in patients with poor-risk MDS/AML (myelodysplastic syndrome/acute myeloid leukemia; N = 30). Overall, 58% had previous Ven exposure and 63% had TP53 mutation; 15/19 had TP53 multihit state. At a median of +55 days, prophylactic maintenance therapy with Ven (400 mg on days 1-14) and Dec-c (35 mg decitabine and 100 mg cedazuridine tablet on days 1, 3, and 5, or days 1, 2, and 3) was initiated for 8 cycles of 42 days each in 26 of 30 patients (87%; of the remaining patients, 3 relapsed early and 1 withdrew). On maintenance, grade 3/4 neutropenia (96%) occurred although infections were rare (n = 2). No dose-limiting toxicities occurred. The 6-month acute GVHD grade 2 to 4 rate was 13%. The 1-year moderate/severe chronic GVHD rate was 31%. At a median follow-up of 25.1 months (range, 15-33), median overall survival (OS) and progression-free survival (PFS) were not reached. On maintenance, 2-year OS was 77%, PFS was 62%, nonrelapse mortality was 0%, and cumulative incidence of relapse was 38%. Exploratory studies identified 96% of patients had pretransplant next-generation sequencing molecular residual disease (MRD) positivity, favorable survival in those with non-TP53 MRD positivity, and delayed conversion on maintenance in 11 of 18 (61%) with TP53-MRD positivity. Patient-reported outcomes assessed in the first 6 months of maintenance were stable except for emotional function, which significantly improved. This trial was registered at www.ClinicalTrials.gov as NCT03613532.

Adrenal lesions often occur in patients with multiple endocrine neoplasia type 1 (MEN1), mostly adrenal cortical adenomas (ACAs), although the frequency of adrenal cortical carcinomas (ACCs) is higher than in the general population. The coexistence of benign and malignant masses has seldom been documented, leaving open the question of ACC progression from benign forms. We report a comprehensive genetic characterization of three adrenal cortical tumor samples obtained from a familial MEN1 patient, operated for the rapid progression of an initially stable nonfunctional adrenal incidentaloma. Histologically, the tumor consisted of a small ACA contiguous to a large ACC, which subsequently relapsed. Exome sequencing of ACC, ACA and recurrence evidenced a MEN1 loss of heterozygosity (LOH) in ACC but not in ACA, where, however, a second hit driven by alternative mechanisms could not be excluded. The majority of the ACA variants were found to co-occurred in ACC (n = 36/42) and were benign, except for two of unknown significance in KANK1 and REN genes, described as associated with renal cancer. Among variants shared between ACC and its recurrence (n = 69), 11 were Tier III, while 2 affecting TP53 and NF1 genes were pathogenic. Bioinformatic clonal evolution analysis identified one clone - characterized by TP53 and NF1 mutations - absent in ACA but present in ACC and recurrence, as well as 2 clones shared between ACA and ACC but lost in the recurrence. In conclusion, comparative Whole Exome Sequencing (WES) analysis of three adrenal tumors in a MEN1 patient suggests a possible relationship between malignant and benign lesions occurring in MEN1 patients, without, however, demonstrating any causal adenoma-to-carcinoma progression driven by MEN1 LOH. Overall, these data further suggest an increased risk of MEN1 patients to develop adrenocortical malignancy.

Rare but distinct: A systematic review of primary neuroendocrine tumors of the breast according to WHO 2019 guidelines.

Background/Objectives: TP53 mutation or deletion status is important for determining cellular responses to DNA-damaging drugs. Oxaliplatin (OXA) is combined with the fluoropyrimidine (FP) drug 5-fluorouracil (5-FU) in the FOLFOX regimen used to treat advanced colorectal cancer (CRC). However, the effects of TP53 deletion on 5-FU + OXA synergy are not well known. We investigated potential synergy between OXA and 5-FU and compared it with OXA synergy with a novel polymeric FP, CF10, in four cell lines harboring either wild-type (WT) or TP53-null status. Methods: Using CompuSyn and the highest single agent (HSA) models, we compared synergy between CF10 and OXA (COXA) and between 5-FU and OXA (FOXA). Cell cycle analysis was performed, as was Western blot quantification of canonical DNA damage pathway proteins. Likewise, immunofluorescent and confocal analysis allowed us to compare topoisomerase 1 cleavage complex and double-strand DNA break formation. Results: COXA synergy displayed minimal TP53 dependence with greatly improved potency compared to FOXA. COXA synergy resulted from OXA increasing: (i) Topoisomerase 1 (Top1) cleavage complex formation; (ii) DNA double-strand breaks (DSBs), and (iii) Checkpoint Kinase 1 and 2 (p-Chk1/2) phosphorylation, consistent with increased replication stress. Additionally, increased S-phase entry in TP53-null cells enhanced synergy between CF10, 5-FU, and OXA as S-phase drugs. Conclusions: Our results demonstrate that OXA synergizes with CF10 more effectively than with 5-FU through enhanced replication stress in both WT and TP53-null cells by causing greater Top1-mediated DNA double-strand breaks. Our studies provide a foundation for further testing of this combination in an orthotopic liver metastatic setting and eventual clinical development.

Objective: To investigate the clinicopathological and molecular genetic features of micronodular thymoma (MNT) and micronodular thymic carcinoma (MNC) with lymphoid stroma. Methods: Seventeen cases of micronodular neoplasms with lymphoid stroma diagnosed at the Third Affiliated Hospital of Soochow University, Changzhou, China from March 2017 to October 2024 were collected (16 cases of MNT and 1 case of MNC). Their clinicopathological data were reviewed, and their paraffin-embedded sections were stained with immunohistochemistry (IHC). Three cases of MNT and one case of MNC were also examined using next generation sequencing (NGS). Results: Of the 17 patients with MNT or MNC, 7 were male and 10 were female, age 66 (63, 72)years old. Microscopically, tumor cells were scattered in the lymphoid stroma as solid epithelioid nests. The tumor nests were relatively regular in 16 cases, and the tumor cells were mainly short spindle or oval, with bland morphology. Tumor cells of MNC showed marked cytological atypia, conspicuous nucleoli, frequent mitotic figures, and fibrous stroma. IHC showed that tumor cells of all 17 cases diffusely expressed CKpan, CK19 and CK5/6. CD5, CD117 and Glut1 were diffusely positive in the MNC case, but negative in any of the 16 MNT cases. In contrast, there were a considerable number of immature T lymphocytes positive for CD99, TdT and CD1a in the lymphoid stroma around MNT tumor nodules, but not in the MNC. Among the 3 MNT cases subjected to NGS testing, 2 cases had missense mutations in GTF2I (p.L424H), and 1 had missense mutations in HRAS (p.G13V and p.L120P). In the MNC case subjected to NGS testing, gene alterations such as a frameshift mutation in BRCA2 (p.D1451Ifs*12) and a missense mutation in TP53 (p.I195T) were detected, but not GTF2I gene mutations. Conclusions: MNC cells are more atypical than MNT cells. CD5, CD117 and Glut1 are diffusely expressed in MNC, but not in MNT. The genetic alterations in MNC are more diverse than those of MNT, but MNC lacks the characteristic GTF2I mutation of MNT.

TP53 mutations are associated with poor prognosis in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Allogeneic hematopoietic stem cell transplantation (allo-HSCT) offers a potential cure, but outcomes are suboptimal and risk stratification remains challenging.This multi-center, retrospective study analyzed 66 patients with TP53-mutated AML/MDS who underwent allo-HSCT. The study endpoints included overall survival (OS), progression-free survival (PFS), and cumulative incidence of relapse (CIR), non-relapse mortality (NRM), and Graft-versus-host disease-free, relapse-free survival (GRFS).After median 1054-day follow-up, 3-year rates for OS, PFS, CIR, NRM, and GRFS were 47.2%, 39.7%, 37.3%, 23%, and 37.4%, respectively. Survival did not differ between AML and MDS. Univariate analysis showed that < 2 somatic myeloid co-mutations predicted inferior OS (3-y OS: 32% vs. 65.9%, p = 0.02) and PFS (27.6% vs. 59.1%, p = 0.01). Age > 50 years adversely affected PFS, and complex karyotype showed a negative trend. Multivariate analysis found no independent factors, likely due to sample size and collinearity. A combined risk factor analysis revealed that patients with ≥ 1 adverse factor (either co-mutations < 2 or complex karyotype) had significantly worse OS (3-y OS: 72.6% vs. 37.2%, p = 0.04) and PFS (3-y PFS: 67.7% vs. 28.9%, p = 0.02) compared to those with neither risk factor.In patients with TP53-mutated AML/MDS undergoing allo-HSCT, a low myeloid co-mutation burden (< 2) is strongly associated with poor outcomes. A composite model integrating co-mutation burden with karyotype may assist in post-transplant risk stratification, offering a practical supplementary parameter when TP53 allelic status is uncertain. This finding requires validation in larger prospective studies.

Abstract Lung cancer in young patients shows a rising incidence and distinct disease biology but remains understudied. We obtained clinical information and somatic mutation data for two large non‐small‐cell lung cancer (NSCLC) patient cohorts from the cBioPortal database—the China cohort ( N = 1948) and the MSK cohort ( N = 6220). Additionally, we extracted clinical data from an internal, real‐world NSCLC patients (GZSY cohort, N = 3914) spanning the past decade. By comparing clinicopathological and genomic features across age and ethnicities, we systematically characterized the molecular and clinical phenotype of early‐onset NSCLC. Young patients were more likely to be female and never smokers, and carried a higher frequency of actionable driver gene mutations, particularly involving EGFR , ALK , ROS1 , and ERBB2 genes. In the China cohort, young patients exhibited significantly higher frequencies of EGFR and ERBB2 mutations and a higher rate of concurrent EGFR ‐ TP53 co‐mutations. Advanced stage at diagnosis and TP53 mutation status emerged as independent adverse prognostic factors in young patients. Integration of multi‐ethnic genomic and clinical data in this study delineates the distinct clinical and molecular landscape of early‐onset NSCLC and supports development of age‐targeted, precision therapeutic strategies.

Clonal haematopoiesis of indeterminate potential (CH), somatic genetic mutations conferring stem cells selective advantage, are associated with increased inflammatory cytokine production, cardiovascular disease (CVD), and malignancy. We investigated whether CH was related to risk of rheumatoid arthritis (RA), an inflammatory autoimmune disease. Within the UK Biobank, we studied 186,577 unrelated individuals with baseline data collection, genome-wide genotyping, whole exome sequencing (WES) read for CHIP, and linked general practice (GP) data for identification of incident RA in follow-up. We excluded those with prevalent RA or taking RA medications at baseline. We tested for associations between variant allele fraction (VAF) >2% and >10% for the 8 most common CHIP mutations in the general population, and incident RA, identified by billing code algorithms. Cox regression models estimated hazard ratios (HR, 95% confidence interval) for incident RA. 594 participants developed incident RA over median follow-up 7.1 years (IQR 6.4-8.0); median time to RA was 3.9 years (IQR 2.0-5.6). Incident RA cases were slightly older at enrolment, more likely to be female, have smoked, have higher body mass index (BMI), and have prevalent CVD, and hypercholesterolaemia at baseline. However, there was no difference in the presence of CHIP mutations at baseline; 6.3% vs. 6.4% of those who did and did not develop incident RA in follow-up had any CHIP mutation at >2% VAF. Common CHIP mutations, including TET2, TP53, and SF3B1 mutations, were not associated with risk of incident RA when restricted to those with most complete general outpatient and hospital record follow-up in the UK Biobank.

Genomic landscape, immune microenvironment and survival in male versus female breast cancer.

Introduction. Richter Transformation (RT), the evolution of chronic lymphocytic leukemia (CLL) into an aggressive lymphoma, remains a major therapeutic challenge, particularly in patients previously exposed to targeted agents. This study reports clinical and molecular features, treatment approaches, and outcomes of RT cases treated in our institution, with specific focus on TP53 aberrations.Methods. A retrospective analysis was conducted on 12 consecutive patients, diagnosed with RT following CLL diagnosis (2012–2025), confirmed by histopathology and immunophenotyping. Data collected included demographics, prior CLL therapies, RT subtype, and molecular/genetic profile (IGHV status, del17p, TP53 mutation, complex karyotype). Response to RT therapy was evaluated per Lugano 2014 criteria; survival was estimated from RT diagnosis to death or last follow-up.Results. Median age at RT was 66 years (range 56–86); 11 patients (91.7%) had received prior CLL therapy, including BTK inhibitors in seven cases. Adverse molecular features were frequent: TP53 mutation in 5/12 (41.7%), del17p in 4/12 (33.3%), and complex karyotype in 3/12 (25%). RT histology was DLBCL-type in 8/12 (66.7%), Hodgkin-like in 3/12 (25%), and mixed in 1/12 (8.3%). First-line RT treatment included chemoimmunotherapy (n=6), targeted combinations (n=4), and cellular therapies (n=2). The overall response rate was 41.7% (5/12). Patients with TP53 aberrations showed markedly inferior outcomes, with median overall survival of 5.2 months compared to 13.8 months in wild-type cases. Among deceased or lost to follow-up patients (n=6), all belonged to the DLBCL subgroup with high-risk molecular features.Conclusions. This real-life case series highlights the aggressive nature of RT in heavily pretreated CLL patients, where TP53 aberration remains the strongest adverse prognostic factor. Despite the introduction of targeted therapies, outcomes remain unsatisfactory, supporting the need for early identification of high-risk cases and integration of innovative, non-chemotherapeutic strategies.

Introduction: High risk MDS (HR-MDS) outcome significantly improved after the introduction of hypomethylating treatment, especially 5’-azacitidine (AZA). Despite AZA is now the standard treatment for HR-MDS, only 30-50% of patients respond to treatment. The identification of response/resistance biomarkers is essential to prevent prolonged exposure and toxicities in unresponsive patients.Methods: We used a comprehensive t-NGS, epigenetic (mERBBS), and transcriptomic (RNA-seq) approach to study 10 HR-MDS, prior and post 4-AZA cycles (5 responders, R, with complete remission; 5 non-responders, NR, with progressive disease). Healthy donor’s (HD) BM samples were used as controls (n=5). 55 BM samples, from the GROM-L multicenter study (n=12) and Gimema MDS0205 study (n=43), were used as independent validation cohort.Results: Age, gender, hematological and genetic features at diagnosis (T0) did not differ in the two subgroups. We identified a mean of 2 mutations (range=0–6, VAF&gt;1%), in 8/10 patients at baseline, with TP53 (40%), DNMT3A (30%), TET2 (30%), ASXL1 (20%) and SETBP1 (20%) as the most frequently mutated genes. After AZA (T4), TP53 mutations became undetectable in 2/5 R patients (40%), while remained unchanged in NR. RNA-seq analysis at T0 (R vs HD, NR vs HD, R vs NR) and at T4 (R vs HD, NR vs HD, R vs NR) collectively showed different baseline transcriptional profiles in the two response-groups, particularly involving immune-related genes.Unsupervised analysis of DNA methylation separated the 2 subgroups, with one branch including all R (at T0 and T4) and HD, while the second all NR (at T0 and T4). mERRBS highlighted a DNA methylation gradient at diagnosis, in accordance with response. Indeed, NR was the most perturbed group, and the most hypermethylated. Within the hypermethylated promoters in NR at T0, BCL9L displayed the highest number of DMRs on its 5’-region. Pyrosequencing and qRT-PCR, were used to validate its hypermethylation and downregulation.Treatment with AZA induced a strong epigenetic shift only in R, with a reduction of DMRs (740 at T4 vs 2.732 at T0) and shift into a hypomethylated pattern (84.4%). By contrast, NR maintained at T4 a comparable number of DMRs (13.281 at T4 vs 13.231 at T0) and confirmed the hypermethylation seen at diagnosis (67.7%). Specifically, the methylation level at homeobox genes backed down to normal levels at T4 only in R. Within these DMRs, PRRX2 gene showed the stronger correlation with response. Hypermethylation in NR was confirmed in an independent validation cohort (n=5 R vs 5 NR, p&lt;0.005).Conclusions: Overall, our results identified a different transcriptional and epigenetic profile according to AZA response, including differential expression of immune-related genes and significant hypermethylation of NR at diagnosis, and a marked epigenetic shift after AZA in R. Of note, hypermethylation at BCL9L promoter and at the PRRX2 gene emerged as candidate predictive markers of AZA-response.

Mantle Cell Lymphoma (MCL) is an aggressive malignancy with variable clinical behavior, largely reflecting the underlying molecular heterogeneity. The genomic landscape of MCL encompasses gene mutations with strong prognostic implications (such as TP53, NOTCH1, KTDM2D, etc) and secondary genetic events, such as copy number variations (CNVs), also implicated in the pathogenesis and prognosis of MCL.We extracted and analyzed, by targeted Next Generation Sequencing (tNGS) with a customized panel of 37 genes, the genomic DNA (gDNA) of 73 diagnostic samples of patients with relapsed/refractory MCL that were enrolled in the Fondazione Italiana Linfomi (FIL) Mantle First-BIO study (NCT04882475). CNVs were annotated with CNVkit, while the TP53 mutation was annotated with MuTec2 and validated through Integrative Genomics Viewer (IGV). The statistical analysis was conducted on R v4.1.2, adopting the time to first relapse or progression of disease (POD) as the primary outcome measure, which was previously defined as a threshold of 24 months since MCL diagnosis, enabling us to divide the cohort into early-POD24 and late-POD24. All patients also had available data for correlating CNVs with the presence of TP53 mutation. We detected 18 different alterations (11 amplifications and 7 deletions) associated with MCL. The most recurrent CNVs were Amp 3q26-q28 (BCL6, 49%), followed by Amp 5q35.3 (HNRNPH1, 45%), Del13q14 (RB1, 34%), and Del 11q22.3-q23.2 (32%). When comparing early and late POD groups, Fisher’s exact test identified Del 13q14 (RB1, p= 0.02, 52% early vs 24% late), Del 6q (p= 0.01, 37% early vs 11% late), and Del 9p21.3 (CDKN2A, p= 0.02, 33% early vs 11% late) more prevalently associated with early POD. Among these, the Del 9p21.3 (CDKN2A) resulted as the stronger predictor of shorter time to POD (p=0.01), independently of TP53 mutation in multivariable analysis. Unsupervised clustering identified molecularly defined clusters associated with significantly different time to POD (p= 0.01). Pairwise log-rank tests confirmed TP53 (mutated vs wild-type) as the strongest prognostic factor, with cluster assessment that improved the prognostic predictivity among patients: clusters TP53-mut vs TP53-WT, p= 0.001, HR=3.92; and p= 0.014, HR= 2.23, respectively (Figure 1).In conclusion, CNV-based molecular clusters might represent a novel approach to identify patients at higher risk of treatment failure, contributing to the prognostic predictivity of TP53 mutation.Acknowledgments. We thank FIL for research funding (Premio Giovani Ricercatori, Ed. 2019) and for supporting our work, the European Union - Next Generation EU - and Ministero della Salute - PNRR-TR1-2023-12378287 - Rafforzamento e potenziamento della ricerca biomedica del SSN (CUP: E13C24000610007).